Download Reconstitution of Outer Membrane Protein Assembly from Purified

Reconstitution of Outer Membrane Protein
Assembly from Purified Components
Christine L. Hagan,1* Seokhee Kim,1* Daniel Kahne1,2†
b-barrel membrane proteins in Gram-negative bacteria, mitochondria, and chloroplasts are assembled
by highly conserved multi-protein complexes. The mechanism by which these molecular machines
fold and insert their substrates is poorly understood. It has not been possible to dissect the folding
and insertion pathway because the process has not been reproduced in a biochemical system.
We purified the components that fold and insert Escherichia coli outer membrane proteins and
reconstituted b-barrel protein assembly in proteoliposomes using the enzymatic activity of a protein
substrate to report on its folding state. The assembly of this protein occurred without an energy source
but required a soluble chaperone in addition to the multi-protein assembly complex.
he outer membranes of Gram-negative
bacteria and the mitochondria and chloroplasts of higher eukaryotes contain proteins with b-barrel structure, which are assembled
in their respective membranes by multi-protein
machines (1–6). The folding and insertion of these
b-barrels must be coordinated because they would
have many unsatisfied hydrogen bonds in the
membrane if they were inserted in an unfolded
state, but conversely, they would be “inside out”
if they folded first in the aqueous environment
and were subsequently inserted. In order to understand how b-barrel proteins assemble into membranes, we purified the proteins comprising the
Escherichia coli outer membrane protein (OMP)–
folding machinery and established a reconstituted
system to monitor the activity of this machinery.
The b-barrel assembly machine (Bam) in
E. coli consists of an integral b-barrel protein,
BamA (formerly YaeT), and four lipoproteins,
BamB, -C, -D, and -E (formerly YfgL, NlpB,
YfiO, and SmpA, respectively). Only BamA and
BamD are essential for cell survival, but deleting
or depleting any member of the complex causes
defects in OMP assembly (6–9). BamA has homologs in prokaryotes and eukaryotes and contains
five periplasmic polypeptide transport–associated
(POTRA) domains, which scaffold the Bam lipoproteins (2–5, 10). Unfolded OMPs are delivered
to this complex after their synthesis in the cytoplasm and translocation across the inner membrane by the secretion machinery (SecYEG) (Fig.
1A) (11). The chaperones, SurA, Skp, and DegP,
prevent unfolded OMPs that are released from the
Sec machine from aggregating and misfolding as
they transit the periplasm. These chaperones are
thought to transport OMPs in two parallel but
separate pathways: one that relies on SurA and
one that involves both Skp and DegP (12, 13).
Most OMPs can be handled by either pathway,
but SurA delivers the bulk of OMPs to the OM
(14).
T
1
Department of Chemistry and Chemical Biology, Harvard
University, Cambridge, MA 02138, USA. 2Department of
Biological Chemistry and Molecular Pharmacology, Harvard
Medical School, Boston, MA 02115, USA.
*These authors contributed equally to this work.
†To whom correspondence should be addressed. E-mail:
[email protected]
890
The process of OMP assembly can be monitored in isolated mitochondria (15) and in vivo
in E. coli (16). We sought to develop an in vitro
system to study the function of the Bam proteins.
Expressing all five bam genes in a single strain
produced a mixture of complexes and subcomplexes that could not be easily separated. Previous lipoprotein and POTRA domain–deletion
experiments indicated that BamA binds BamB
independently from BamCDE (8, 10); thus,
we expressed the subcomplexes BamAB and
BamCDE separately and reconstructed the full
complex in vitro (17). The reconstructed complex
was identical to the native complex, as analyzed
with blue native polyacrylamide gel electrophoresis (BN-PAGE) (Fig. 1B). The purified Bam
complex had an apparent molecular weight that
is too small to accommodate more than one copy
of BamA, and the ratio of BamA:B:C:D was
1:1:1:1 (fig. S1). Either one or two copies of
BamE may have been present; its small size prevents a definitive conclusion. The Bam complex may exist as a higher-order oligomer in
the membrane in vivo, but the relative stoichiometry is expected to be as we have determined.
Purified Bam complex was incorporated
into liposomes, as others have done to reconstitute complexes that handle membrane proteins (18–22). To follow OMP assembly in the
proteoliposomes, we used a substrate OMP that
possesses enzymatic activity, OmpT. OmpT is a
b-barrel outer membrane protease that cleaves
peptides between two consecutive basic residues,
and its activity can be monitored by using a
fluorogenic peptide (23). Urea-denatured OmpT
was incubated with the periplasmic chaperone
A
B
Outer
Membrane
Folding and
Insertion
BamA
BN-PAGE
1
2
SDS-PAGE
100-
669-
-BamA
75440-
B
amE
E
BamE
B
amD
D
BamD
B C
BamC
BamB
B
50-
232-
Recognition and Binding
140Periplasm
37-
-BamB
-BamC
2520-
-BamD
15-
SurA, Skp,
DegP
-BamE
10-
Chaperone Transport
500
N
BamA
400
300
B
D
E
C
200
Inner
Membrane
SecYEG
Cytoplasm
N
100
Translocation
0
0
5
10
15
20
Elution Volume (mL)
25
Ribosomal Synthesis
signal
sequence
C
C
-BamA
-BamA
250
600
BamA
-BamC
500
400
300
-BamD
200
100
200
-BamB
B
150
100
50
0
0
5
10
15
20
Elution Volume (mL)
0
25
-BamE
BamA
D
0
E
C
5
10
15
20
Elution Volume (mL)
25
Fig. 1. The OMP assembly pathway and the purified Bam complex and subcomplexes. (A) The E. coli OMP
biogenesis pathway. (B) Blue native gel analysis, SDS-PAGE, and gel filtration chromatogram of the
purified Bam complex. The complex isolated from cells expressing the complex at basal levels (lane 1) and
the overproduced and reconstructed complex (lane 2). (C) SDS-PAGE and gel filtration chromatograms of
the purified two- and four-protein Bam subcomplexes.
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SCIENCE
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REPORTS
REPORTS
A
7500
7500
5000
5000
2500
2500
0
0
0
B
[SurA]
0 µM
5 µM
10 µM
15 µM
20 µM
25 µM
30 µM
40 µM
50 µM
60 µM
80 µM
30
100 µM
BamABCDE
No Complex
10
20
Time (min)
30
0
10
20
Time (min)
C
300
SurA n-OmpT
OmpT + n SurA
BamABCDE
200
Misfolding/Aggregation
SurA n-OmpT-BamABCDE
100
n SurA +
Folded OmpT
BamABCDE +
LPS, Peptide
0
0
25
50
75
[SurA] (µM)
100
Cleaved Fluorescent Peptide
Fig. 2. SurA and the Bam complex facilitate OmpT assembly in proteoliposomes. (A) Preincubated
solutions of urea-denatured OmpT with SurA were diluted [at time (t) = 0 min] into (left) liposomes or
(right) proteoliposomes that contain the Bam complex. OmpT was present in each reaction at a final
concentration of 10 mM, and the concentration of SurA was varied from 0 to 100 mM. (B) The amount of
active OmpT (reflected in the slope from 15 to 30 min) in the reactions in (A) as a function of SurA
concentration in the presence (black triangles) or absence (red squares) of the Bam complex. (C)
Schematic of the reconstitution reaction pathway.
A
10000
10075-
7500
5000
No Complex 50BamAB
BamACDE 37BamABCDE
2500
25-
0
-BamA
BamB
BamC
-BamD
20-
0
10
20
Time (min)
30
B
-BamE
15-
C
100
400
75
300
50
200
25
100
0
0
10
20
Time (min)
30
0
0
10
20
Time (min)
30
Fig. 3. OmpT assembly requires specific components of the Bam complex. (A) Fluorescence
produced in the presence of the Bam complex and subcomplexes (left). OmpT and SurA are diluted
to final concentrations of 10 mM and 100 mM, respectively. SDS-PAGE of these proteoliposomes
indicates that they contain equal amounts of the complexes (right). (B) Eight different experiments
were normalized to their maximum fluorescence values and then averaged. The error bars
represent the SD among these experiments. (C) The gradient of the fluorescence data in (A).
Fig. 4. OmpT assembled
by the Bam complex is
folded on SDS-PAGE and
resistant to membrane extraction. Shown are pellets
of the reactions of SurA
and 35S-labeled OmpT with
proteoliposomes containing the Bam complexes. 35S-labeled OmpT and SurA were diluted to final concentrations of 0.4 mM and
100 mM, respectively. Extraction in 100 mM sodium carbonate results in a threefold enrichment of the
folded material relative to the unfolded material.
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VOL 328
SurA, and this chaperone-OmpT complex was
diluted into solutions containing the proteoliposomes, the fluorogenic peptide, and lipopolysaccharide (LPS), which is required for OmpT
activity (17). OmpT assembly was observed as an
increase in the rate of fluorescence production.
OmpT activity increased with the concentration of SurA and in the presence of the Bam
complex (Fig. 2A and figs. S2 and S4). OmpT
activity saturated at the same SurA concentration in the presence or absence of the complex,
which suggests that SurA plays a general role in
delivering the unfolded protein in a state that can
be folded and also that the Bam complex handles
OmpT bound to SurA (Fig. 2, B and C). The concentrations of SurA at which we saw a substantial
improvement in OmpT assembly (above 20 mM)
are comparable with the reported binding constants for SurA and model peptides (1 to 14 mM)
(24, 25). OmpT contains several putative SurAbinding sites (26, 27), and the sigmoidal relationship may indicate that multiple SurA molecules
are involved in generating a SurAn-OmpT complex that can be folded efficiently.
We then wanted to determine whether the assembly observed in the presence of the Bam complex reflected the function of the complex and not
a nonspecific property of the proteoliposomes
(28, 29). We compared the activity of the fiveprotein Bam complex with subcomplexes containing two (BamAB) and four proteins (BamACDE),
which had comparable stability as judged by their
behavior on gel filtration chromatography (Fig. 1C).
The components of these subcomplexes were incorporated into proteoliposomes in equal proportion to those of the five-protein complex (Fig. 3A).
Cleavage of the fluorogenic substrate was greatly
reduced in proteoliposomes containing the subcomplexes as compared with those containing the fiveprotein complex (Fig. 3A and fig. S3). The activity
of the four-protein complex was always less than
that of the five-protein complex (Fig. 3B). In the first
five minutes of the experiment, OmpT assembly
was orders of magnitude faster in the presence of the
five-protein complex (Fig. 3C). These large differences in activity at early time points are relevant
given that in vivo pulse-chase experiments indicate
that OMP assembly occurs in the cell on a similar
time scale of 30 s to several minutes (30, 31). The
four-protein complex appeared to have some
activity on this time scale, which BamB dramatically improved. Although there was a background
rate of OmpT folding in the absence of the Bam
complex, the Bam proteins significantly improved
the kinetics of OmpT assembly.
In order to verify that increased fluorescence
correlated with increased amounts of folded
OmpT, we ran the products of our folding reactions on SDS-PAGE gels without and with prior
heat denaturation. b-barrel proteins remain folded
on SDS-PAGE if they are not boiled and consequently run faster than their unfolded forms.
Purified 35S-labeled OmpT was incubated with
SurA and then diluted into proteoliposomes, as in
the fluorescence experiments. After 30 min, the
14 MAY 2010
891
REPORTS
reaction solutions were centrifuged and the pellets were run on SDS-PAGE (Fig. 4 and fig. S5).
Much more folded OmpT was observed in the
reaction of SurAn-OmpT with the full Bam
complex than in the reactions with the subcomplexes. The yield of folded protein represented by
the faster migrating band was about 7%, as determined by five separate experiments. The folded
OmpT was resistant to extraction from the pellet
by incubation in 100 mM sodium carbonate for
30 min, suggesting that the folded protein had
been integrated into the membrane.
The nonessential lipoprotein BamB is important in the assembly of OMP substrates delivered
by SurA; the five-protein complex had significantly higher activity than the four-protein complex,
BamACDE. Thus, some or all of the BamCDE
proteins are required for OmpT assembly, but
BamB appears to have a specific function that,
when coordinated with the other components, increases the activity of the complex. The functional
importance of both SurA and BamB is consistent
with in vivo observations that these proteins play
related but not redundant roles in OMP biogenesis.
Deleting surA or bamB produces identical phenotypes with respect to the kinetics of conversion of
unfolded mature LamB to folded monomers (31).
In addition, simultaneous deletion of the surA and
bamB genes results in a phenotype that is severely
defective in the assembly of OMPs and much
sicker than either single-deletion strain (32, 33).
Our results also suggest that SurA and BamB
both affect the efficiency of OMP assembly. Our
highly simplified system thus appears to recapitulate elements of the cellular process and clearly
demonstrates that a nonessential protein can alter
the activity of essential proteins to regulate the
efficiency of the process that they catalyze.
We do not know if the Bam complex in proteoliposomes can process multiple substrate molecules, but clearly the Bam complex can assemble a
b-barrel protein in the membrane without an input
of energy. In contrast, the inner membrane secretion machinery couples energy-driven translocation
to insertion (11). Although it remains possible that
b-barrel assembly may be coupled to an energydriven process in the cell, it is remarkable that these
six proteins (the Bam complex and a chaperone)
are sufficient to perform the folding and insertion
process. Together, they perform a chemical transformation that we do not understand, and that they
are able to do so without energy suggests that their
structure inherently facilitates OMP assembly.
References and Notes
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from Amherst College.
Supporting Online Material
www.sciencemag.org/cgi/content/full/science.1188919/DC1
Materials and Methods
Figure S1 to S6
Tables S1 and S2
1 March 2009; accepted 29 March 2009
Published online 8 April 2009;
10.1126/science.1188919
Include this information when citing this paper.
Additive Genetic Breeding Values
Correlate with the Load of Partially
Deleterious Mutations
Joseph L. Tomkins,1* Marissa A. Penrose,1 Johan Greeff,1,2 Natasha R. LeBas1
The mutation-selection–balance model predicts most additive genetic variation to arise from
numerous mildly deleterious mutations of small effect. Correspondingly, “good genes” models of
sexual selection and recent models for the evolution of sex are built on the assumption that
mutational loads and breeding values for fitness-related traits are correlated. In support of this
concept, inbreeding depression was negatively genetically correlated with breeding values for traits
under natural and sexual selection in the weevil Callosobruchus maculatus. The correlations were
stronger in males and strongest for condition. These results confirm the role of existing, partially
recessive mutations in maintaining additive genetic variation in outbred populations, reveal the
nature of good genes under sexual selection, and show how sexual selection can offset the cost of sex.
artially recessive, deleterious mutations of
small effect are thought to be responsible
for much of the observed additive genetic
variation in traits that are closely associated with
fitness (1–4). These mutations are also central to
“good genes” models of sexual selection, which
posit that the additive genetic breeding values of
males must correlate negatively with mutational
load (5–8). The contribution of nonadditive allelic
effects to additive genetic variation through
mutation-selection balance has empirical support
from mutation-accumulation experiments (9) and
molecular-evolution surveys (10), but not from
recent biometric analyses (11–13).
P
1
Centre for Evolutionary Biology, School of Animal Biology,
The University of Western Australia, Nedlands, WA 6009,
Australia. 2Department of Agriculture and Food, 3 Baron-Hay
Court, South Perth, WA 6151, Australia.
*To whom correspondence should be addressed. E-mail:
[email protected]
14 MAY 2010
VOL 328
SCIENCE
Here we develop Fox’s proposal (14) for detecting among-family variation in inbreeding depression. Inbreeding depression is the reduction
in fitness observed in the offspring of related
(compared with unrelated) parents (4, 15) and is
believed to be predominantly caused by the increased homozygosity of numerous, at least partially recessive, deleterious mutations of small
effect (4, 15). By estimating the correlation between the load of mutations carried by a pair of
parents and their breeding value, we were able to
test the central assumption of good genes models
of sexual selection and contribute to our understanding of the maintenance of genetic variation.
We used a seven-generation pedigree of
cow-pea weevils Callosobruchus maculatus
(Coleoptera: Bruchidae) reared on black-eye beans
(Vigna unguiculata) to estimate predicted breeding
values, heritabilities, and inbreeding depression
in a range of life-history (LH) and morphological
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Reconstitution of Outer Membrane Protein Assembly from Purified
Components
Christine L. Hagan et al.
Science 328, 890 (2010);
DOI: 10.1126/science.1188919
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